EP1384999A1 - Bakteriotoxinadsorptionsmittel und screening-verfahren dafür - Google Patents

Bakteriotoxinadsorptionsmittel und screening-verfahren dafür Download PDF

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EP1384999A1
EP1384999A1 EP02714580A EP02714580A EP1384999A1 EP 1384999 A1 EP1384999 A1 EP 1384999A1 EP 02714580 A EP02714580 A EP 02714580A EP 02714580 A EP02714580 A EP 02714580A EP 1384999 A1 EP1384999 A1 EP 1384999A1
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Prior art keywords
lys
gln
val
glu
ser
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French (fr)
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EP1384999A4 (de
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Koichi Fukase
Hans Peter Nestler
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Japan Science and Technology Agency
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Japan Science and Technology Corp
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the present invention relates to a method for screening and preparing an endotoxin adsorbent and to an adsorbent having a specific structure found by the method.
  • Lipopolysaccharide is a main component for surface layer of cells of Gram-negative bacteria such as Escherichia coli and salmonella. It acts as a signal for invasion of bacteria and produces various mediators such as cytokine, prostaglandin, lipid such as PAF, or NO to activate the immune system.
  • LPS endotoxin
  • cytokine cytokine
  • prostaglandin lipid
  • lipid such as PAF, or NO
  • an advantageous immune response takes place while, when lipopolysaccharide excessively acts due to infectious diseases or the like, the immune system runs out of control and high fever, systemic blood coagulation, etc.
  • Toraymyxine (trade name; Toray), where polymyxin B, an antibiotic, is immobilized with fiber which is used for an endotoxin adsorption therapy, that has been developed and actually used as an adsorbent for lipopolysaccharide.
  • the invention has been achieved in view of the above-mentioned current situation and its object is to provide a novel lipopolysaccharide adsorbent (endotoxin adsorbent) which can be easily synthesized, can be easily adjusted various analogous structures easily, and can be used for adsorption of various kinds of lipopolysaccharides, and a method for screening the same.
  • a novel lipopolysaccharide adsorbent endotoxin adsorbent
  • the invention relates to a method for screening an adsorbent for endotoxin characterized in that a binding assay with a peptide library is conducted using a lipid A radioisotope-labeled preparation where radioactive element is introduced into a phosphonoxyethyl derivative of a lipid A of a biosynthetic precursor type or a lipid A of an Escherichia coli type and also to an adsorbent for endotoxin which is found by the screening method.
  • the invention also relates to a peptide library where a solid phase carrier having a functional group is bound to a compound, having a functional group which is able to bind to the said functional group and having 2 or more other functional groups, and is further bound to an oligopeptide.
  • the invention further relates to a peptide library where a solid carrier having a functional group is bound to a compound, having a functional group which is able to bind to the said functional group and having 2 or more other functional groups, and is further bound to a compound, having a functional group which is able to bind to the said other functional groups and having 2 or more still other functional groups and, after that, an oligopeptide is bound thereto.
  • the invention still further relates to a lipid A radioisotope-labeled preparation where radioactive element is introduced into a phosphonoxyethyl derivative of a lipid A of a synthetic precursor type or a lipid A of an Escherichia coli type.
  • Fig. 1 shows the result where a binding ability of the peptide library of the invention (using Tenta Gel which is a polyethylene glycol-polystyrene resin as a solid phase carrier) to lipid A, lipid A analog and lipopolysaccharide was investigated.
  • Fig. 1 (a) is a result of the binding assay with a phosphonoxyethyl derivative of an Escherichia coli type lipid A (PE 506), (b) is that with lipid A of an Escherichia coli type (506), (c) is that with a phosphonoxyethyl derivative of lipid A of a biosynthetic precursor type (PE 406), (d) is that with lipid A of a biosynthetic precursor type (406), (e) is that with lipopolysaccharide of an Escherichia coli Re mutant (ReLPS) and (f) is that with an Escherichia coli lipopolysaccharide (LPS).
  • Fig. 2 shows the result where a binding ability of the peptide library of the invention (using TOYOPEARL which is a hydrophilic vinyl polymer as a solid phase carrier) to lipid A, lipid A analog and lipopolysaccharide was investigated.
  • FIG. 2 (a) is a result of the binding assay with lipid A of an Escherichia coli type (506), (b) is that with a lipopolysaccharide of an Escherichia coli Re mutant (ReLPS) and (c) is that with an Escherichia coli lipopolysaccharide (LPS).
  • ReLPS lipopolysaccharide of an Escherichia coli Re mutant
  • LPS Escherichia coli lipopolysaccharide
  • Tritium ( 3 H) is exemplified as a radioactive element in a radioisotope-labeled lipid A analog where radioactive element is introduced into a phosphonoxyethyl derivative of lipid A of a biosynthetic precursor type or lipid A of an Escherichia coli type used in the invention.
  • the peptide library used in the invention includes a peptide library which is prepared by binding a solid phase carrier or a soluble carrier having a functional group to a compound having a functional group which is able to bind to the said functional group and also having 2 or more other functional groups followed by binding an oligopeptide thereto.
  • the solid phase carrier or the soluble carrier having a functional group includes a solid phase carrier or a soluble carrier having an amino group.
  • the compound, having a functional group which is able to bind to the amino group and also having 2 or more other functional groups includes a basic amino acid represented by the formula HOOC(NH 2 )CH(CH 2 ) n NH 2 (in the formula, n is an integer of 1 to 4), and a carboxylic acid having 2 or more hydroxyl groups such as deoxycholic acid and chenodeoxycholic acid.
  • the solid phase carrier or the soluble carrier having a functional group is a solid phase carrier or a soluble carrier having an amino group
  • the compound, having a functional group which is able to bind to the said amino group and also having two or more other functional groups is an amino acid represented by the formula HOOC(NH 2 )CH(CH 2 ) n NH 2 (in the formula, n is an integer of 1 to 4)
  • the compound, having a functional group which is able to bind to the said other functional groups and having two or more still other functional groups includes a basic amino acid represented by the formula HOOC(NH 2 )CH(CH 2 ) n NH 2 (in the formula, n is an integer of 1 to 4).
  • an oligopeptide having 2 to 20 amino acid residues, preferably 3 to 10, more preferably, 4 to 8 may be exemplified as an oligopeptide which is to be bound to the solid phase carrier or the soluble carrier having a functional group via a compound having a functional group which is able to bind to the said functional group or having two or more other functional groups, or via a compound where the solid phase carrier or the soluble carrier having a functional group is bound to a compound having a functional group which is able to be bound to the said functional group and two or more other functional groups and further bound to a compound having a functional group which is able to be bound to the said other functional groups and having two or more still other functional groups.
  • amino acid residue there is no particular limitation for the type of the amino acid residue, there may be exemplified Gly, D,L-Val, D,L-Phe, D,L-Pro, D,L-Ser, D,L-Gln, D,L-Glu, D,L-Lys, etc. as preferred ones.
  • a specific example for the peptide library concerning the invention includes a compound represented by the following formula [1], [2] or [3].
  • a spherical part represents a solid phase carrier or a soluble carrier, and AA 1 , AA 2 , AA 3 and AA 4 each independently represents an amino acid residue.
  • a spherical part represents a solid phase carrier or a soluble carrier, and AA 1 , AA 2 , AA 3 and AA 4 each independently represents an amino acid residue.
  • a spherical part represents a solid phase carrier or a soluble carrier, and AA 1 , AA 2 , AA 3 and AA 4 each independently represents an amino acid residue.
  • peptide library represented by the above formula [1], [2] or [3] there is no particular limitation for the type of the amino acid residue represented by AA 1 , AA 2 , AA 3 and AA 4 , and any amino acid residue may be used.
  • the examples of the preferred ones are Gly, D,L-Val, D,L-Phe, D,L-Pro, D,L-Ser, D,L-Gln, D,L-Glu and D,L-Lys.
  • the endotoxin adsorbent of the invention can be detected and extracted by means of a binding assay to the above peptide library using the above radioisotope-labeled lipid A preparation.
  • the specific examples of the endotoxin adsorbent of the invention includes such as those where AA 1 , AA 2 , AA 3 and AA 4 in the formula [1] comprise any of the combinations mentioned in the following Table 1 AA1 AA2 AA3 AA4 D ⁇ Phe D ⁇ Val L ⁇ Gln D ⁇ Phe L ⁇ Lys D ⁇ Ser D ⁇ Val D ⁇ Gln D ⁇ Pro D ⁇ Phe L ⁇ Phe L ⁇ Val D ⁇ Gln D ⁇ Gln L ⁇ Phe D ⁇ Lys D ⁇ Val L ⁇ Pro D ⁇ Ser D ⁇ Ser those where AA 1 , AA 2 , AA 3 and AA 4 in the formula [2] comprise any of the combinations mentioned in the following Table 2.
  • AA1 AA2 AA3 AA4 D ⁇ Pro L ⁇ Glu D ⁇ Glu L ⁇ Val D ⁇ Val D ⁇ Pro D ⁇ Lys L ⁇ Gln D ⁇ Val L ⁇ Glu L ⁇ Val D ⁇ Glu L ⁇ Val D ⁇ Pro D ⁇ Val D ⁇ Ser L ⁇ Val D ⁇ Pro L ⁇ Phe Gly D ⁇ Lys L ⁇ Glu D ⁇ Glu L ⁇ Gln L ⁇ Lys D ⁇ Lys D ⁇ Ser L ⁇ Phe L ⁇ Lys Gly L ⁇ Ser D ⁇ Gln D ⁇ Glu D ⁇ Ser L ⁇ Glu ,or those where AA 1 , AA 2 , AA 3 and AA 4 in the formula [3] comprise any of the combinations mentioned in the following Table 3.
  • the solid phase carrier or the soluble carrier having a functional group used in the invention includes, for example, resin or polyethylene glycol having functional group such as amino group, carboxyl group and hydroxyl group and, preferably, resin in beads.
  • resin examples of such resin are a polyethylene glycol-polystyrene resin having the above-mentioned functional group [examples of the specific trade name are Tenta Gel S-NH 2 (Rapp Polymere), Aminomethyl Nova Gel HL (Novabiochem), etc.], a hydrophilic vinyl polymer [examples of the specific trade name are TSK gel AF-Amino TOYOPEARL 650 S (Tosoh)], etc.].
  • an encode molecular tweezers library (peptide library) was prepared by a split synthesis.
  • chenodeoxycholic acid was used as a template and tetrapeptide library was bound to two hydroxyl groups via Gly residue (diarm L1), tetrapeptide library was bound to each of two amino groups of L-Lys (diarm L2) and L-Lys was bound to each of amino groups of L-Lys and tetrapeptide library was bound to 4 amino groups (tetraarm L3).
  • Tenta Gel which was a polyethylene glycol-polystyrene resin which was swollen in water was used taking the fact that an assay will be conducted in water into consideration.
  • L1 deoxycholic acid was first introduced into a solid phase carrier and, after that, Fmoc-Gly was bound to two hydroxyl groups.
  • Lysine (L2) and trilysine (L3) cores were prepared by a common peptide solid phase synthetic method.
  • L2 and L3 cores were prepared by a common peptide solid phase synthetic method.
  • 15 kinds of amino acids Gly, D and L-Val, Phe, Ser, Gln, Glu, and Lys and Pro
  • the radioisotope-labeled lipid A analog represented by the above formula [4] (hereinafter, abbreviated as [ 3 H]PE 506) or the radioisotope-labeled lipid A analog represented by the above formula [5] (hereinafter, abbreviated as [ 3 H]PE 406) was used and a binding assay to a molecular tweezers library was carried out.
  • a binding assay to a molecular tweezers library using a radioisotope-labeled compound Nestler established it already and the means was applied (Nestler H. P.; Wennemers H.; Sherlock R.; Dong D.L.-Y Bioorg. Med. Chem Lett ., 6, 1327 (1996).).
  • the binding assays were carried out to phosphonoxyethyl derivatives of lipid A of an Escherichia coli type (hereinafter, abbreviated as PE 506), lipid A of an Escherichia coli type (hereinafter, abbreviated as 506), phosphonoxyethyl derivative of lipid A of a biosynthetic precursor type (hereinafter, abbreviated as PE 406), lipid A of a biosynthetic precursor type (hereinafter, abbreviated as 406), lipopolysaccharide of Re mutant of Escherichia coli (ReLPS) and lipopolysaccharide of Escherichia coli ( E. coli O111:B4 LPS (Sigma)).
  • ReLPS has a structure where two residues of the sugar called Kdo are bound to a lipid A moiety lacking in most. of core polysaccharide and
  • the Limulus activity test is a method where using the reaction that lipopolysaccharide activates a coagulation enzyme of LAL (Limulus Amebocyte Lysate) which is a component extracted from blood corpuscles of horseshoe crab (Limulus polyphemus , etc.), endotoxin activity is subjected to a colorimetric determination.
  • LAL Liulus Amebocyte Lysate
  • Binding assays to 6-Tenta Gel, 11-Tenta Gel, 12-Tenta Gel, 16-Tenta Gel and 17-Tenta Gel was carried out same as above using PE 506, 506, PE 406, 406, ReLPS and Escherichia coli lipopolysaccharide ( E. coli O111:B4 LPS (Sigma)) and Limulus activity of the supernatant liquid was measured. When such an endotoxin was adsorbed with the resin, the observed absorbance lowers. In addition, since each resin was washed with water before conducting the assay here, it is likely that most of the amino groups on the resin were in a free state.
  • Tenta Gel is a graft polymer of polyethylene glycol with polystyrene and, although it is swollen in water, it is a resin having a high hydrophobic property.
  • the Escherichia coli lipopolysaccharide (LPS) has a long sugar chain and it was presumed that the hydrophilic sugar chain disturbed permeation into the resin.
  • TSK gel AF-Amino TOYOPEARL 650 S having a high hydrophilic property as a carrier
  • 6-Toyopearl, 11-Toyopearl, 12-Toyopearl, 16-Toyopearl and 17-Toyopearl to which molecular tweezers 6, 11, 12, 16 and 17 were bound
  • ReLPS and Escherichia coli lipopolysaccharide E. coli O111:B4 LPS (Sigma)
  • ReLPS and Escherichia coli lipopolysaccharide E. coli O111:B4 LPS (Sigma)
  • the adsorbent of the invention uses a natural substance (natural amino acid), which is a big advantage.
  • Kiesel gel 60 (E. Merck; 0.040 to 0.063 mm) was used for the silica gel column chromatography.
  • Sephadex LH-20 was purchased from Pharmacia Biotech, Sweden.
  • Anhydrous CH 2 Cl 2 and CHCl 3 were prepared by distillation using calcium hydride as a dehydrating agent.
  • Anhydrous tetrahydrofuran (THF) and anhydrous benzene were purchased from Kanto Kagaku K. K. NaB 3 H 4 (specific activity: 222 GBq mmol -1 ) was purchased from Amersham Life Science.
  • an imaging plate BAS-TR 2040S (Fuji Photo-Film) was used and, for the detection, a bio-imaging analyzer BAS-1500 MAC (Fuji Photo-Film) was used.
  • the organic layer was washed with an aqueous solution of Na 2 S 2 O 3 (2 ⁇ 50 mL) and a saturated saline solution (20 mL), dried over Na 2 SO 4 and concentrated in vacuo to obtain a diol as a crude product (458 mg).
  • the diol was dissolved in anhydrous benzene (10 mL) and lead tetraacetate (Pb(OAc) 4 ) (purity: 90%, 119 mg, 242 mmol) was added thereto followed by stirring for 30 minutes.
  • the reaction mixture was charged to a silica gel column (3 g) and eluted with ethyl acetate.
  • N,N-Diethyl-1,5-dihydro-3 H -2,4,3-benzodioxaphosphepin-3-amine (89 mg, 0.37 mmol) and tetrazole (25 mg, 0.32 mmol) were added at 0°C to a solution of the compound 21* (144 mg, 59.9 mmol) in CH 2 Cl 2 (14 mL). After the reaction mixture was stirred at room temperature for 30 minutes, it was cooled at -78°C. mCPBA (70%, 81 mg, 0.37 mmol) was added thereto followed by stirring for 45 minutes. The reaction was stopped by adding a saturated Na 2 S 2 O 3 followed by extracting with CHCl 3 .
  • Pd-black 130 mg was added to a solution of the compound 22* (57 mg, 22 mmol) in THF (8 mL) and a catalytic reduction was carried under pressure (7 kg cm -2 ) at room temperature for 2 hours. Triethylamine was added to neutralize and the Pd-black was removed by filtration.
  • the resulting compound was identical with an authentic sample of non-labeled 4 on a chromatography.
  • the organic layer was washed with an aqueous solution of Na 2 S 2 O 3 (2 ⁇ 50 mL) and a saturated saline solution (20 mL), dried over Na 2 SO 4 and concentrated in vacuo to obtain a diol as a crude product (458 mg).
  • the diol was dissolved in anhydrous benzene (10 mL) and lead tetraacetate (Pb(OAc) 4 ) (purity: 90%, 68 mg, 140 mmol) was added thereto followed by stirring for 30 minutes.
  • the reaction mixture was charged to a silica gel column (3 g) and eluted with ethyl acetate.
  • Pd-black 120 mg was added to a solution of the compound 26* (17 mg, 7.8 mmol) in THF (6 mL) and a catalytic reduction was carried under pressure (7 kg cm -2 ) at room temperature for 2 hours. Triethylamine was added to neutralize and the Pd-black was removed by filtration.
  • the resulting compound was identical with an authentic sample of non-labeled 5 on a chromatography.
  • An encode molecular tweezers library was prepared by a split-mix synthetic method.
  • As a solid carrier 15 g of Tenta Gel S-NH 2 (Rapp Polymere; particle size: 90 ⁇ m; 0.29 mmol/g of NH 2 ) were used.
  • a peptide chain was elongated using a standard Fmoc/Boc (fluorenylmethoxycarbonyl/butyloxycarbonyl) peptide solid-phase synthetic method.
  • Fmoc/Boc fluorenylmethoxycarbonyl/butyloxycarbonyl
  • HOBt/DIC hydroxybenzotriazole/diisopropyl carbodiimide
  • Catechol Tag was introduced by an amide bond formation reaction in about 1% of the amino groups by an HOBt/DIC method via a carboxyl group.
  • chenodeoxycholic acid (L1) was used as a core structure
  • deoxycholic acid was firstly introduced into a solid phase carrier and 6 equivalents of Fmoc-Gly-F, 6 equivalents of triethylamine and 0.06 equivalent of dimethylaminopyridine were made to act with two hydroxyl groups in DMF whereby Fmoc-Gly was bound.
  • Lysine (L2) and trilysine (L3) cores were prepared by a common peptide solid phase synthetic method.
  • [ 3 H] PE 506 (62 GBq/mmol, 1.4 mg, 47 MBq) was dissolved in water (2 ml). A molecular tweezers library (15.7 mg, about 19,000 beads) was suspended in water (200 ⁇ l) and a [ 3 H] PE 506 solution (100 ⁇ l, 2.4 MBq) was added. The suspension of [ 3 H] PE 506 (130 ⁇ M) and resin was slowly shaken at 4°C for 41 hours. An operation where the resin was precipitated by centrifugation and the supernatant liquid was removed was repeated three times.
  • Glass plate was treated with an NaCIO solution, well washed with water, taken out after dipping in 0.5 gelatin solution and dried with air to give a glass plate on which gelatin was coated.
  • the above resin was placed thereon and dried with air. This was covered with a Kodak autoradiography emulsion which was previously melted by warming and, after confirming that it was well solidified, it was allowed to stand under shading at -78°C for 12 days. After the glass plate was dipped in a developing liquid for 10 minutes, it was washed with water. After being dipped in a fixing solution for 15 minutes, it was washed with water. Surroundings of the resin having radioactivity became black by adhesion of silver to gelatin.
  • the radioactive resin was taken out and Tag bound to the resin was cut from the resin, detected by an electron capture gas chromatography (ECGC) and decoded to find molecular tweezers binding to 19 kinds of [ 3 H] PE 506.
  • ECGC electron capture gas chromatography
  • [ 3 H] PE 406 (64 GBq/mmol, 0.6 mg, 26 MBq) was dissolved in water (1 ml).
  • a molecular tweezers library (24.2 mg, about 29,000 beads) was suspended in water (200 ⁇ l) and a [ 3 H] PE 406 solution (100 ⁇ l, 2.6 MBq) was added.
  • the suspension of [ 3 H] PE 406 (130 ⁇ M) and resin was slowly shaken at 4°C for 48 hours. The resin was precipitated by centrifugation and the supernatant liquid was removed. An operation where 1 ⁇ l of water is added to the resin, the resulting mixture is shaken, the resin is precipitated by centrifugation and the supernatant liquid is removed was repeated three times.
  • Resin was placed on a gelatin-coated glass plate and dried with air. This was covered with a Kodak autoradiography emulsion which was previously melted by warming and, after confirming that it was well solidified, it was allowed to stand under shading at -78°C for 7 days. The radioactive resin was taken out and subjected to an ECGC analysis. A molecular tweezers binding to 32 kinds of [ 3 H] PE 406 was found.
  • Tenta Gel S-NH 2 (Rapp Polymere; particle size: 90 ⁇ m; 0.29 mmol/g of NH 2 ) or TSK gel AF-Amino TOYOPEARL 650 S (Tosoh; 0.1 mmol/ml) was used.
  • Peptide chain was elongated using a standard Fmoc/Boc (fluorenylmethoxycarbonyl/butyoxycarbonyl) peptide solid phase synthetic method.
  • As a peptide condensation method an HOBt/DIC (hydroxybenzotriazole/diisopropylcarbodiimide) was used. The end point of the peptide condensation reaction was confirmed by disappearance of blue color by a bromophenol blue test.
  • the resin was washed with DMF (4.0 ml) twice and a DMF solution (3 ml) of Boc-L-Lys(Boc)-OH (104 mg, 0.300 mmol), a DMF solution (1 ml) of diisopropylcarbodiimide (DIC) (47.0 ⁇ l, 0.300 mmol) and a DMF solution (1 ml) of 1-hydroxybenzotriazole (HOBt) (40.5 mg, 0.300 mmol) were added thereto followed by stirring at room temperature for 2 hours. After filtration, the resin was washed with DMF (4.0 ml) four times. A peptide condensation reaction using Boc-L-Lys(Boc)-OH was repeated once again.
  • DIC diisopropylcarbodiimide
  • HOBt 1-hydroxybenzotriazole
  • a DMF solution (3 ml) of Fmoc-L-Lys(Boc)-OH (281 mg, 0.600 mmol), a DMF solution (1 ml) of DIC (94.0 ⁇ l, 0.600 mmol) and a DMF solution of HOBt (81.1 mg, 0.600 mmol) were added followed by shaking at room temperature for 2 hours. After filtration, the resin was washed with DMF (4.0 ml) for four times. A peptide condensation reaction using Fmoc-L-Lys(Boc)-OH was repeated once again. Unreacted amino groups were similarly blocked using acetic anhydride.
  • Boc group was removed by the same manner as in the above (2).
  • Fmoc group was removed by the same manner as in the above (4).
  • Fmoc group was removed by the same manner as in the above (4).
  • Fmoc group was removed by the same manner as in the above (4).
  • PE 506 (0.06 mg) was dissolved in water (0.80 ml). The molecular tweezers 6 to 17 Tenta Gel (about 20 mg) were suspended in water (100 ⁇ l) and a PE 506 solution (25 ⁇ l) was added thereto. The suspension was slowly shaken overnight at room temperature. The resin was precipitated by centrifugation and the supernatant liquid was used for a TLC analysis and a Limulus activity test.
  • Binding assays were similarly carried out using 506 (0.56 mg), PE 406 (0.7 mg), 406 (0.7 mg), PeLPS (0.85 mg) and LPS (1 mg) and the resulting supernatant liquids were used for the measurement of Limulus activity (in the case of 506, it was used for a TLC analysis as well).
  • the result of the TLC analysis is as shown in Table 6.
  • A a solution where 40 mg of sodium nitrite was dissolved in 4 ml of concentrated hydrochloric acid and 96 ml of water
  • the invention relates to methods for screening and preparation of an endotoxin adsorbent and to an adsorbent having a specific structure which was found by that methods.
  • the advantages are that it provides a novel endotoxin adsorbent where synthesis is easy, various analogous structures can be easily prepared and can be used for an adsorbent of various endotoxins.
  • the adsorbent of the invention has a big advantage of using a natural product (natural amino acid).

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  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Endocrinology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Saccharide Compounds (AREA)
EP02714580A 2001-04-18 2002-04-16 Bakteriotoxinadsorptionsmittel und screening-verfahren dafür Withdrawn EP1384999A4 (de)

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JP2001119547 2001-04-18
JP2001119547A JP3893030B2 (ja) 2001-04-18 2001-04-18 細菌内毒素吸着剤及びそのスクリーニング方法
PCT/JP2002/003757 WO2002086503A1 (fr) 2001-04-18 2002-04-16 Adsorbants de bacteriotoxines et procede de criblage desdits adsorbants

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DE102016205950A1 (de) 2016-04-08 2017-10-12 Dietrich Seidel Mittel zur Verwendung bei entzündlichen Zuständen der Schleimhäute

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US7799347B2 (en) 2006-09-07 2010-09-21 Chondrex Inc. Endotoxin-adsorbent for the prevention and treatment of autoimmune diseases
JP5718574B2 (ja) * 2010-01-29 2015-05-13 学校法人同志社 ペプチドのスクリーニング方法

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Publication number Priority date Publication date Assignee Title
DE102016205950A1 (de) 2016-04-08 2017-10-12 Dietrich Seidel Mittel zur Verwendung bei entzündlichen Zuständen der Schleimhäute

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CA2444055A1 (en) 2002-10-31
JP2002311029A (ja) 2002-10-23
US20040137543A1 (en) 2004-07-15
JP3893030B2 (ja) 2007-03-14
EP1384999A4 (de) 2008-05-21

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